The Cellular And Molecular Mechanisms Of HBV Immune Tolerance By WT-HBs-Tg Parabiotic Mice Model

Chronic hepatitis B virus(HBV)infection is one of the highest risks in the development of hepatocellular carcinoma(HCC).In 2019,approximately 296 million people have been chronically infected with HBV in the world,among which about 820,000 deaths due to chronic HBV infection.It is well known that HBV-induced immune tolerance accounts for the chronic infection in hosts.Researches on HBV mouse models demonstrated that central tolerance to HBV existed in HBsAg(hepatitis B surface antigen)-transgenic(Tg)mice(HBs-Tg mice);however,the immune response to HBV vaccine could be inspired in adult HBs-Tg mice after boosting with potent adjuvants,leaving a mystery to explore its immune tolerance.However,owing to the limitations of the HBV mouse models,further studies of the regulatory mechanisms of the HBV immune tolerance were hindered.Thus,a model to observe how the immune system naturally responds to HBV and how to influence HBV immune tolerance is necessary.In this study,we developed a new mouse model for HBV immune tolerance research by conjoining WT(donor)mouse and HBs-Tg(host)mouse via parabiotic surgery,in order to see how immunocompetent WT mice naturally respond to HBV,and how tolerant HBs-Tg mice influence the anti-HBV immunity from WT mice.It was found that the proportion and number of CD8~+T cells were significantly increased,and their proliferative ability and cytotoxicity were markedly enhanced,causing inflammatory injury in the liver of HBs-Tg parabionts.More than 60%of CD8~+T cells were derived from donor WT mice in the liver of HBs-Tg parabionts.After 4 weeks of parabiosis,HBsAg-specific CD8~+T cells were detected in the liver of HBs-Tg parabionts,which were derived from donor WT mice,but not host HBs-Tg mice.Notably,when HBs-Tg mice but not WT mice were performed splenectomy,the frequency of HBsAg-specific CD8~+T cells was decreased and the positive rate was reduced from 100%to 50%in the parabiotic model.These results indicated that the spleen of HBs-Tg mouse was indispensable for the generation of HBsAg-specific CD8~+T cells,while the spleen of WT mouse was not in the WT-HBs-Tg parabiotic model.We also found that no HBsAg-specific CD8~+T cells were detected in the liver of HBsTg parabionts when donor CD4~+T cells were deficient.However,when host CD4~+T cells of HBs-Tg were deficient,donor HBsAg-specific CD8~+T cells were still generated in the liver of Cd4-/-HBs-Tg after parabiosis with WT mice.These results indicated that the priming of donor HBsAg-specific CD8~+T cells was dependent on donor CD4~+T cells rather than host CD4~+T cells in the HBs-Tg parabionts.Compared with donor CD4~+T cells,host CD4~+T cells harbored a higher frequency of regulatory T cells(Tregs),with higher expressions of inhibitory receptors.Moreover,it was found that host Tregs from HBs-Tg mice inhibited CD8~+T cell proliferation involving inhibitory receptor TIGIT(T cell immunoglobulin and immune receptor tyrosine-based inhibitory motif domain)in vitro.However,the frequency of donor IFN-γ+ Th1 cells and the ratio of total Th1/Tregs were markedly increased in the spleen of HBs-Tg parabionts,indicating that increased donor IFN-γ+ Th1 cells were able to overcome the inhibition of Tregs in the spleen of HBs-Tg parabionts,thereby effectively helping donor CD8~+T cells to generate anti-HBV-specific immune response.Further,compared with the control HBs-Tg mice,the number of liver tumor nodules was significantly reduced and the development of HCC was markedly slowed in HBs-Tg parabionts after 12-13 months of parabiosis.There were no significant differences in the proportions and expression levels of inhibitory receptors of CD8~+T and CD4~+T cells in the liver of HBs-Tg parabionts compared with the control HBs-Tg mice,indicating that they all showed exhausted phenotypes.In conclusion,by using WT-HBs-Tg parabiotic mice model the major mechanisms underlying immune tolerance in HBs-Tg mice were revealed,including incompetent anti-HBV CD8~+T cells and insufficient help from CD4~+T cells,which accounted for chronic HBV persistence.Moreover,the development of HCC was slowed in HBs-Tg parabionts after parabiosis with WT mice.These results provide a theoretical basis for reversing HBV immune tolerance and inducing effective HBV-specific CD8~+T cell responses,and further immunotherapy strategies for chronic HBV infection.